Alzheimer's disease (AD) is characterised by memory loss, insidious cognitive decline, profound neurodegeneration, and the extracellular accumulation of amyloid-beta (Aβ) peptide in senile plaques and intracellular accumulation of tau in neurofibrillary tangles. Synaptic dysfunction and loss is the strongest pathological correlate of cognitive decline in AD with increasing evidence implicating neuropathological forms of both amyloid-beta and tau protein in this process. A large amount of evidence suggests that oligomeric forms of Aβ, associated with senile plaques, are toxic to synapses but the precise localisation of Aβ and which forms are synaptotoxic remain unknown. Using the high-resolution technique, array tomography, this thesis characterised the synaptic localisation of different forms of Aβ oligomers in a mouse model of amyloidopathy. These results show that different oligomeric Aβ species are present in both presynapses and postsynapses. This study highlights the potential of array tomography for rapid testing of aggregation state specific Aβ antibodies in brain tissue. Following these results, the presence of tau at synapses was examined. Despite the knowledge that tau spreads through defined synaptic circuits, it is currently unknown whether synapse loss occurs before the accumulation of tau or as a consequence. To address this, array tomography was used to examine a mouse model in which mutant P301L human tau is expressed primarily in the entorhinal cortex (rTgTauEC). It has previously been shown that rTgTauEC mice exhibit neuronal loss in the entorhinal cortex and synapse density loss in the middle molecular layer (MML) of the dentate gyrus at 24 months of age. The density of tau-expressing and total presynapses, and the spread of tau into the postsynapse in the MML of 3-6, 9, and 18 month old mice were examined. No loss of synapse density was observed in the MML up to 18 months of age, even in axons expressing tau. Despite the maintenance of synapse density, we see spread of human tau from presynaptic terminals to postsynaptic compartments in the MML at very early ages. This indicates that the spread of tau through neural circuits is not due to the degeneration of axon terminals and is an early feature of the disease process. Following examination of both synaptic amyloid-beta and tau in separate models, this thesis then examined how these two proteins may be synergistically working together to drive synaptic pathology. To investigate this a novel mouse model was used in which amyloid-beta deposits are present in combination with non-mutated human tau expression (APP/PS1 + hTau). These results suggested that the addition of human tau expression does not increase plaque associated synapse loss, neither does it increase the proportion of synapses colocalising with amyloid-beta. Similarly the presence of human tau at individual postsynapses was not enhanced in the presence of oligomeric Aβ. Surprisingly, intact long-term recognition memory was observed in APP/PS1 + hTau mice. However a hyperactive phenotype was detected in these mice that could be prevented upon tau suppression. This suggests a synergistic relationship may exist in the presentation of this phenotype. Finally in the last part of this thesis, synapses from post-mortem human Alzheimer's disease and age-matched controls were investigated. It has previously been suggested that both amyloid-beta and tau can interfere with mitochondrial transport to the synapse and mitochondrial function. For this reason the presence of synaptic mitochondria at both the presynapse and postsynapse was determined in order to investigate any alteration in the diseased state. A reduction in the proportion of presynapses with multiple mitochondria present was detected in anterior/posterior transverse temporal cortex (BA41/42). This was not observed in dorsolateral prefrontal cortex (BA46), suggesting either a selective vulnerability of the former brain region or a selective resistance of the latter brain region, to mitochondrial depletion at the synapse. The findings presented in this thesis demonstrate that when investigated in isolation, pathological forms of amyloid-beta are present at a subset of synapses where they may contribute to toxicity, whilst the spread of tau protein is an early feature of the disease process and occurs prior to overt synapse loss. This thesis also explores the proposed synergistic relationship between amyloid-beta and tau using a novel mouse model and human post-mortem brain tissue. Since these two proteins both have been implicated in synaptic dysfunction, investigating Aβ and tau in new mouse models and human brain tissue will be instrumental in furthering our understanding of mechanisms and features of synaptotoxicity that could be important therapeutic targets.

<div><b>Rationale:</b> Focal cortical dysplasia (FCD) is a neurodevelopmental disorder that is associated with abnormal cortical development and is one of the most common drug-resistant epilepsies. The mechanistic target of rapamycin (mTOR) pathway is a highly complex pathway </div><div>associated with cell proliferation, synaptic plasticity, neuroinflammation, and cortical development. Hyperactivation of this pathway has also been implicated in hyperexcitability, seizures, and accumulation of beta-amyloid (Aβ) plaques and neurofibrillary tangles (NFT) through hyperphosphorylation of tau. Interestingly, Aβ and hyperphosphorylated tau have been reported in both rodent models and human patients of temporal lobe epilepsy (TLE) and FCD however, the mechanisms through which this occurs are still yet to be defined. Therefore, to identify the possible link between Aβ and tau pathology in FCD, we determined the spatial distribution and protein levels of Aβ and phosphorylated tau (p-tau) along with mTOR signaling </div><div>molecules. We hypothesized that there would be presence of Aβ and tau pathology as well as an increase in Aβ and p-tau protein levels that would be correlated with hyperactivation of the mTOR and GSK3 signaling pathways in tissue biopsies from human FCD patients compared to brain tissues from non-epileptic (NE) individuals.</div><div><br></div><div><b>Methods:</b> Cortical brain samples surgically resected from patients with FCD were used and compared to NE samples surgically resected from glioblastoma patients with no history of seizures or epilepsy. Immunostaining was used to determine the distribution of phosphorylation of S6 (p-S6), a marker for mTOR activation, and NeuN, a marker for neurons, along with Aβ and p-tau. Additionally, western blotting (WB) was used to determine the levels of mTOR signaling through p-S6 and GSK3 (p-GSK) along with Aβ and p-tau.</div><div><br></div><div><b>Results:</b> We found cortical dyslamination, mTOR activation, p-tau, and Aβ accumulation in cortices of patients with FCD with drug-resistant epilepsy. However, we did not find a </div><div>significant difference in the protein levels of p-S6 (p = 0.422), p-GSK3 (p = 0.947), p-tau (p = 0.649), and Aβ (p = 0.852) in cortical tissue homogenates derived from FCD patients when compared to those of NE samples. Additionally, we did not find sex differences in the protein </div><div>levels of p-S6 (p = 0.401), p-GSK3 (p = 0.331), p-tau (p = 0.935), and Aβ (p = 0.526). There was no significant correlation between age and p-S6 (p = 0.920), age and p-GSK3 (p = 0.089), age and p-tau (p = 0.956), and age and Aβ (p = 0.889). Moreover, there was no significant correlation between mTOR activation (p-S6), Aβ (p = 0.586) and p-tau (p = 0.059) nor GSK3 activation (p-GSK3), Aβ (p = 0.326), and p-tau (p = 0.715). Lastly, there was no significant correlation within the mTOR and GSK3 pathway activation within the same patients (p = 0.602).</div><div><br></div><div><b>Conclusion:</b> These data suggest that mTOR hyperactivation occurs alongside the presence of Aβ and tau pathology. However, several unknown factors such as medical and medication history may be altering the expression or suppression of these proteins. Additionally, there may be alternative pathways that crosstalk with mTOR signaling therefore influencing Aβ and tau pathology in FCD patients with drug-resistant epilepsy. Further investigation will need to be conducted to understand the detailed mechanisms through which Aβ and tau pathology occur in </div><div>FCD.</div>